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Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
Multiple Pseudomonas species secrete exolysin-like toxins and provoke Caspase-1-dependent macrophage death
Pauline Basso1, Pierre Wallet2, Sylvie Elsen1
1CNRS-ERL5261, INSERM, U1036, CEA, Bacterial Pathogenesis and Cellular Responses, Biosciences and Biotechnology Institute of Grenoble, University Grenoble Alpes, France.
Abstract:
Pathogenic bacteria secrete protein toxins that provoke apoptosis or necrosis of eukaryotic cells. Here, we developed a live-imaging method, based on incorporation of a DNA-intercalating dye into membrane-damaged host cells, to study the kinetics of primary bone marrow-derived macrophages (BMDMs) mortality induced by opportunistic pathogen Pseudomonas aeruginosa expressing either Type III Secretion System (T3SS) toxins or the pore-forming toxin, Exolysin (ExlA). We found that ExlA promotes the activation of Caspase-1 and maturation of interleukin-1β. BMDMs deficient for Caspase-1 and Caspase-11 were resistant to ExlA-induced death. Furthermore, by using KO BMDMs, we determined that the upstream NLRP3/ASC complex leads to the Caspase-1 activation. We also demonstrated that Pseudomonas putida and Pseudomonas protegens and the Drosophila pathogen Pseudomonas entomophila, which naturally express ExlA-like toxins, are cytotoxic toward macrophages and provoke the same type of pro-inflammatory death as does ExlA+ P. aeruginosa. These results demonstrate that ExlA-like toxins of two-partner secretion systems from diverse Pseudomonas species activate the NLRP3 inflammasome and provoke inflammatory pyroptotic death of macrophages.
Insights
Pseudomonas Exolysin (ExlA) toxins trigger pyroptotic death in macrophages by activating Caspase-1 and the NLRP3 inflammasome. This inflammatory cell death mechanism is conserved across diverse Pseudomonas species expressing ExlA-like toxins.
Area of Science:
- Microbiology
- Immunology
- Cell Biology
Background:
- Pathogenic bacteria employ protein toxins to induce host cell death, impacting infection outcomes.
- Understanding the mechanisms of bacterial toxin-induced cell death is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the kinetics of macrophage death induced by Pseudomonas aeruginosa toxins.
- To elucidate the molecular pathways involved in Exolysin (ExlA)-mediated cell death.
Main Methods:
- Developed a live-imaging method using DNA-intercalating dyes to track membrane-damaged host cells.
- Utilized knockout (KO) bone marrow-derived macrophages (BMDMs) deficient in Caspase-1, Caspase-11, and NLRP3/ASC components.
- Assessed cytotoxicity of various Pseudomonas species expressing ExlA-like toxins.
Main Results:
- Exolysin (ExlA) induced Caspase-1 activation and interleukin-1β maturation in BMDMs.
- Mice deficient in Caspase-1 and Caspase-11 exhibited resistance to ExlA-induced death.
- The NLRP3 inflammasome complex was identified as upstream of Caspase-1 activation.
- ExlA-like toxins from Pseudomonas putida, Pseudomonas protegens, and Pseudomonas entomophila induced similar pro-inflammatory macrophage death.
Conclusions:
- ExlA-like toxins from diverse Pseudomonas species activate the NLRP3 inflammasome, leading to pyroptotic death in macrophages.
- This mechanism of inflammatory cell death is conserved across different Pseudomonas species.
- The findings provide insights into bacterial pathogenesis and host immune responses.
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